Signaling through G Protein Receptors

Question 1

1. Draw the membrane topology a G protein-coupled receptor and identify the basic structural characteristics that mediate ligand binding and coupling to G proteins.
2. Explain how G protein-coupled receptors activate hetero-trimeric G proteins and diagram the GTP-hydrolysis cycle of G protein signaling.
3. Describe the function of second messengers in receptor signaling and give two examples for how they are generated by activated G proteins.
4. Explain how receptor activation leads to signal termination through receptor desensitization and coupling to additional pathways.
5. Give two examples of drugs that act through modulating different steps in a receptor-G protein-second messenger signaling cascade.

Answer 1

x

Question 2

G protein coupled receptors have ____ transmembrane domains arranged into a barrel-like pore where the ligand binds. The amino terminus extends into the ___ while the carboxyl terminus extends into the ______. Loops that extended into the cytoplasm (_____) confer specificity for the G protein binding

Answer 2

7; ECF; cytoplasm; i-loops

Question 3

Effector regulation is mediated by both free _____ and _____.

Answer 3

Alpha-GTP and Beta-Gamma subunits.

Question 4

How are G proteins activated?

Answer 4

Binding of an agonist to the G protein coupled receptor (GPCR) triggers transfer of a GTP to the G-alpha subunit, replacing a GDP. The G-alpha is active when bound to GTP and can signal induce effector molecules.

Question 5

How are G proteins deactivated?

Answer 5

The G-alpha subunit is a GTPase, which cleaves the GTP to GDP, inactivating the signaling pathway [Nucleotide Hydrolysis (GTPase)].

Question 6

The CTX Cholera toxin affects CL- channels, which apparently are GPCR channels. How does this toxin work?

Answer 6

CTX: Cholera Toxin-ADP ribosylates G-alphas near the GTP binding site of the alpha subunit to inhibit GTPase activity thus converting the G protein to the active state even without receptor activation. [No deactivation]

Question 7

How does the Bordella Pertussis (whooping cough) toxin work?

Answer 7

PTX: Pertussis Toxin-ADP ribosylates G-alpha i/o family near the C- terminus of alpha subunit to lock the G protein heterotrimer in the inactive state by preventing receptor coupling. [No activation]

Question 8

What are the signaling molecules of the sympathetic nervous system? Differentiate between synaptic messenger and hormonal. What is the receptor?

Answer 8

1) norepinephrine (NE) released at synapses
2) epinephrine (adrenaline) released into blood by the adrenal glands

Adrenergic receptor

Question 9

What is the signaling molecule of the parasympathetic branch? What is the receptor?

Answer 9

1) acetylcholine (Ach) released at synapses

2) muscarinic cholinergic receptor

Question 10

The activation reaction (exchange of GTP for GDP) can also be catalyzed with the assistance of a ______.

Once the alpha unit is bound to GTP it ______ and both are available to act on effector proteins. The alpha unit has an intrinsic GTPase activity that eventually hydrolyzes the GTP to GDP (this can be assisted by ________). Once the alpha unit is again associated with GDP, it can be recycled back to the membrane and G protein coupled receptor.

Answer 10

guanine nucleotide exchange factor (GEF)

dissociates from the beta-gamma unit; GTPase activating proteins or GAPs

Question 11

In the peripheral vasculature, Alpha1 adrenergic receptors bind _____ which causes the exchange of GDP for GTP on Gq alpha unit. Gq-GTP interacts with the ____ molecule which cleaves minor membrane lipid _____. This causes an increase in the second messengers ___ and ___ both which eventually stimulate ______ resulting in _____ of smooth muscles.

Answer 11

Norepinephrine; PLC (phospholipase C); PIP2; DAG and IP3; Ca channels; contraction

[peripheral vasoconstriction increases bp, shifts focus to heart, lungs. Flight/fight]

Question 12

In the heart Beta adrenergic receptors bind ____ causing GDP/GTP exchange and the activation of the Gs alpha unit which binds to ______. This ___ it. AC creates ____, a second messenger, increasing levels in the cell, which in turn activate _____. _____ phosphorylates Ca channels, leading to influx of Ca. These steps lead to _____.

Answer 12

Norepinephrine;  
adenylyl cyclase (AC); activates; 
cAMP; 
protein kinase A (PKA); 
PKA; 
contraction of cardiac muscle

Question 13

Again in the heart, lets look at an m2 muscarinic cholinergic receptor. It receives parasympathetic input when acetylcholine binds the receptor, activating the ____ with GTP which binds AC and _____ leading to _____.

Answer 13

Gi protein; prevents an increase in cAMP; decreased heart rate

Question 14

In a given target tissue, what two GCPR types would likely not be found together? Why?

Answer 14

Alpha 1 adrenergic and m3 muscarinic cholinergic receptors. Why? Both have Gq internal sequences and induce similar pathways. This would cause problems since parasympathetic and sympathetic innervation would cause the same result.

Question 15

Gs =

Answer 15

Stimulatory G protein

Question 16

Gi=

Answer 16

Inhibitory G protein

Question 17

Gq = ?

Answer 17

acts on PLPc (Phospholipase C)

Question 18

What effect would metaprolol (B1 selective antagonist) have on heart rate?

Answer 18

decrease it [and subsequently blood pressure]

Question 19

What effect would prazocin (Alpha 1 selective antagonist) have on bp?

Answer 19

decrease bp [block vasoconstriction response in periphery]

Question 20

Phosphodiesterases degrade _____ into _____, which renders it unable to ______

Answer 20

cAMP; AMP; stimulate PKA

[cGMP-specific PDE inhibitors: Viagra (PDE5)]

[caffeine is a PDE inhibitor, which prevents downregulation of the pathway]

Question 21

The Beta 1 Adrenergic receptor receives what neurotransmitter? Agonist binding does what?

Answer 21

Norepinephrine. Stimulates adenylyl cyclase(AC) cAMP production

Question 22

The Alpha 1 Adrenergic receptor receives what neurotransmitter? Agonist binding does what?

Answer 22

Norepinephrine. Stimulates phospholipase C (PLC) Ca2+ and lipid signals.

Question 23

The M2 muscarinic receptor receives what neurotransmitter? Agonist binding does what?

Answer 23

Acetylcholine. Inhibits adenylyl cyclase(AC) cAMP production.

Question 24

The M3 muscarinic receptor receives what neurotransmitter? Agonist binding does what?

Answer 24

Acetylcholine. Stimulates phospholipase C (PLC) Ca2+ and lipid signals.

Question 25

What two G proteins compete (symp/parasymp)? Describe the battle.

Answer 25

Binding of Ach to the M2 muscarinic receptor stimulates Gi-alpha which inactivates Adenylyl Cyclase. Binding of NE to the Beta1 adrenergic receptor releases Gs-alpha which stimulates AC. Whichever signal is stronger wins.

[Both of these G proteins (Gi and Gs) can be active at the same time! Both can bind AC, and whichever has the more dominant signallying “wins” and effects heart rate more.]

Question 26

Talk about m2-muscarinic cholinergic receptor signaling by K+ channel activation in heart.

Answer 26

Finally we get to use the Gamma subunit. This binds GIRK, a G activated potassium channel. Binding by the gamma subunit opens the channel and potassium flows out, leading to membrane hyperpolarization and decreased excitability and a decrease in heart rate and contraction.

Question 27

What is likely to happen through epinephrine binding of Beta-adrenergic receptors in the lung?

Answer 27

Smooth muscle relaxation leading to bronchodilation (and dilation of vasculature supplying blood to lungs,heart and muscle)

[epinephrine–>Gs-alpha–>AC–> increase cAMP–> increase PKA which inhibits smooth muscle contraction]

Question 28

What is likely to happen through Ach binding of m3-muscarinic cholinergic receptors in the lung?

Answer 28

Bronchoconstriction

[Ach–>Gq alpha–>cascade–> Ca2+ release–> contraction]

Question 29

Describe two ways the G signaling pathway can become desensitized.

Answer 29

1) GRK = kinase
GRK phosphorylates the cytoplasmic loops of the G protein coupled receptor–> inactivates it. These phosphorylated residues recruit a protein called beta arrestin. Binding of beta arrestin further prevents G proteins from associating with the receptor, preventing further signaling.

2) Beta arrestin also acts as an adaptor for endocytic machinery, which causes internalization of the entire receptor. Thus, the cell becomes less sensitive to ligand because of fewer receptors. These internalized receptors can then be degraded or re-sensitized and recycled.

Question 30

Metoprolol:

Answer 30

is an antagonist of the beta adrenergic receptor which blocks the activation of the G protein with GTP. Thus, it prevents an increase in cAMP in response to Norepinephrine and lowers heart rate.

Question 31

Prazosin:

Answer 31

is an antagonist of the alpha1 adrenergic receptor which prevent Gq-GTP from interacting with PLC. This in turn prevents the creation of DAG and IP3 receptors.

Question 32

Atropine:

Answer 32

is an acetylcholine antagonist that prevents activation of Gi protein with GTP and thus is an antagonist of parasympathetic signaling. This results in a shift of balance with the Gs protein, whereby Gs “wins” and cAMP levels increase, increasing muscle contraction of the heart.

Question 33

PDE inhibitors – Caffeine, Theophyline, Milinirone, Rolipram, etc:

Answer 33

These target the phosphodiesterases (PDE) of the message pathway to activate signaling. PDEs degrade cAMP by cleaving it to AMP, which is not capable of activating PKA, and thus terminate signaling. Many of these PDEs are constitutively active. By inhibiting the terminator of the signal, PDE inhibitors are able to extend signaling.